RESUMO
BACKGROUND: Ischemic stroke immediately evokes a strong neuro-inflammatory response within the vascular compartment, which contributes to primary infarct development under vessel occlusion as well as further infarct growth despite recanalization, referred to as ischemia/reperfusion injury. Later, in the subacute phase of stroke (beyond day 1 after recanalization), further inflammatory processes within the brain parenchyma follow. Whether this second wave of parenchymal inflammation contributes to an additional/secondary increase in infarct volumes and bears the potential to be pharmacologically targeted remains elusive. We addressed the role of the NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome in the subacute phase of ischemic stroke. METHODS: Focal cerebral ischemia was induced in C57Bl/6 mice by a 30-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with the NLRP3 inhibitor MCC950 therapeutically 24 h after or prophylactically before tMCAO. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 7 after tMCAO. RESULTS: Infarct sizes on day 7 after tMCAO decreased about 35% after delayed and about 60% after prophylactic NLRP3 inhibition compared to vehicle. Functionally, pharmacological inhibition of NLRP3 mitigated the local inflammatory response in the ischemic brain as indicated by reduction of infiltrating immune cells and reactive astrogliosis. CONCLUSIONS: Our results demonstrate that the NLRP3 inflammasome continues to drive neuroinflammation within the subacute stroke phase. NLRP3 inflammasome inhibition leads to a better long-term outcome-even when administered with a delay of 1 day after stroke induction, indicating ongoing inflammation-driven infarct progression. These findings may pave the way for eagerly awaited delayed treatment options in ischemic stroke.
Assuntos
Isquemia Encefálica , Inflamassomos , AVC Isquêmico , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Camundongos , Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Inflamassomos/antagonistas & inibidores , Inflamassomos/metabolismo , Inflamação/complicações , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/complicações , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte-platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2-/-) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα-von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2-/- mice. During ischemia-reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2-/- mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke.
Assuntos
Isquemia Encefálica , Proteína ORAI2 , Acidente Vascular Cerebral , Animais , Plaquetas/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/prevenção & controle , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/metabolismo , Camundongos , Camundongos Knockout , Neuroproteção , Proteína ORAI2/genética , Acidente Vascular Cerebral/metabolismoRESUMO
In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.
Assuntos
Lesões Encefálicas , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Encéfalo/metabolismo , Lesões Encefálicas/metabolismo , Endoglina/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Camundongos , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood-brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.
Assuntos
Apoptose , AVC Isquêmico/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Barreira Hematoencefálica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Inflamação , AVC Isquêmico/genética , AVC Isquêmico/fisiopatologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Neurônios/fisiologia , ProteômicaRESUMO
In ischemic stroke (IS) impairment of the blood-brain barrier (BBB) has an important role in the secondary deterioration of neurological function. BBB disruption is associated with ischemia-induced inflammation, brain edema formation, and hemorrhagic infarct transformation, but the underlying mechanisms are incompletely understood. Dysfunction of endothelial cells (EC) may play a central role in this process. Although neuronal NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome upregulation is an established trigger of inflammation in IS, the contribution of its expression in EC is unclear. We here used brain EC, exposed them to oxygen and glucose deprivation (OGD) in vitro, and analyzed their survival depending on inflammasome inhibition with the NLRP3-specific drug MCC950. During OGD, EC death could significantly be reduced when targeting NLRP3, concomitant with diminished endothelial NLRP3 expression. Furthermore, MCC950 led to reduced levels of Caspase 1 (p20) and activated Gasdermin D as markers for pyroptosis. Moreover, inflammasome inhibition reduced the secretion of pro-inflammatory chemokines, cytokines, and matrix metalloproteinase-9 (MMP9) in EC. In a translational approach, IS was induced in C57Bl/6 mice by 60 mins transient middle cerebral artery occlusion and 23 hours of reperfusion. Stroke volume, functional outcome, the BBB integrity, and-in good agreement with the in vitro results-MMP9 secretion as well as EC survival improved significantly in MCC950-treated mice. In conclusion, our results establish the NLRP3 inflammasome as a critical pathogenic effector of stroke-induced BBB disruption by activating inflammatory signaling cascades and pyroptosis in brain EC.
Assuntos
Barreira Hematoencefálica/metabolismo , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Furanos/administração & dosagem , Indenos/administração & dosagem , Inflamassomos/antagonistas & inibidores , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Substâncias Protetoras/administração & dosagem , Piroptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Glucose/metabolismo , Inflamassomos/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Resultado do TratamentoRESUMO
Patients with atrial fibrillation and previous ischemic stroke (IS) are at increased risk of cerebrovascular events despite anticoagulation. In these patients, treatment with non-vitamin K oral anticoagulants (NOAC) such as edoxaban reduced the probability and severity of further IS without increasing the risk of major bleeding. However, the detailed protective mechanism of edoxaban has not yet been investigated in a model of ischemia/reperfusion injury. Therefore, in the current study we aimed to assess in a clinically relevant setting whether treatment with edoxaban attenuates stroke severity, and whether edoxaban has an impact on the local cerebral inflammatory response and blood-brain barrier (BBB) function after experimental IS in mice. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in male mice receiving edoxaban, phenprocoumon or vehicle. Infarct volumes, functional outcome and the occurrence of intracerebral hemorrhage were assessed. BBB damage and the extent of local inflammatory response were determined. Treatment with edoxaban significantly reduced infarct volumes and improved neurological outcome and BBB function on day 1 and attenuated brain tissue inflammation. In summary, our study provides evidence that edoxaban might exert its protective effect in human IS by modulating different key steps of IS pathophysiology, but further studies are warranted.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Piridinas/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Tiazóis/farmacologia , Animais , Barreira Hematoencefálica/patologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Humanos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Inflamação/patologia , Camundongos , Índice de Gravidade de Doença , Acidente Vascular Cerebral/patologiaRESUMO
OBJECTIVE: Immune responses are an integral part of the complex reactions to acute cerebral ischemia and contribute to infarct expansion and tissue remodeling. Among damage-associated molecular patterns (DAMPs) the high-mobility group box 1 protein (HMGB1) and calprotectin (S100A8/A9) are released from dying cells and activate the innate immune system. METHODS: To assess DAMPs concentrations and related leukocytic infiltration directly and locally in human stroke patients we performed microcatheter sampling from within the core of the occluded vascular compartment before recanalization by mechanical thrombectomy. These samples from the core of a sealed cerebral-ischemic arterial compartment were compared with systemic control samples from the internal carotid artery obtained after recanalization. RESULTS: We found increased plasma levels of total free HMGB1 (+33%) and increased S100A8/A9 (+8%) locally within the ischemic cerebral compartment vs. systemic levels. Local concentrations of HMGB1 were associated with more extensive structural brain infarction on admission. In addition, local ischemic HMGB1 and S100A8/A9 concentrations were associated with the numbers of leukocytes that infiltrate the occluded compartment by collateral pathways. CONCLUSION: This is the first direct human observation of a local increase in DAMPs concentrations in a uniquely sealed vascular compartment of the ischemic cerebral circulation. These data provide an important pathophysiological link between ischemia-induced cell death and stroke-related inflammation.
RESUMO
In acute ischemic stroke due to large vessel occlusion (LVO) infarcts rapidly grow into the penumbra, which represents dysfunctional, but still viable brain tissue amenable to rescue by vessel recanalization. However, infarct progression and/or delayed patient presentation are serious and frequent limitations of this so far only acute therapy. Thus, a major goal of translational research is to "freeze" the penumbra already during LVO (before opening the vessel) and thereby extend individual time windows for non-futile recanalization. We used the filament occlusion model of the middle cerebral artery (MCAO) in mice and assessed progressive infarction under occlusion at 2, 3, and 4 h after onset. We show that blocking the activatory platelet receptor glycoprotein (GP)VI substantially delayed progressive neocortical infarction compared to isotype control antibody treated mice. Moreover, the local vascular recruitment of infiltrating neutrophils and T-cells was mitigated. In conclusion, our experimental data support ongoing clinical trials blocking platelet GPVI in acute ischemic stroke.
Assuntos
Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII-/- mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII-/- mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII-/- mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII-/- mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII-/- mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.
Assuntos
Lesões Encefálicas Traumáticas/genética , Disfunção Cognitiva/genética , Deficiência do Fator XII/genética , Fator XII/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas Traumáticas/sangue , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/patologia , Disfunção Cognitiva/sangue , Disfunção Cognitiva/complicações , Disfunção Cognitiva/patologia , Corticosterona/sangue , Modelos Animais de Doenças , Deficiência do Fator XII/sangue , Deficiência do Fator XII/complicações , Deficiência do Fator XII/patologia , Humanos , Memória/fisiologia , Camundongos , Camundongos Knockout , Agregação Plaquetária/genética , Complexo Glicoproteico GPIb-IX de PlaquetasRESUMO
The clinical and preclinical research of ischemic strokes (IS) is becoming increasingly comprehensive, especially with the emerging evidence of complex thrombotic and inflammatory interactions. Within these, the blood brain barrier (BBB) plays an important role in regulating the cellular interactions at the vascular interface and is therefore the object of many IS-related questions. Consequently, valid, economic and responsible methods to define BBB integrity are necessary. Therefore, we compared the three ex-vivo setups albumin Western blot (WB), IgG WB and albumin intensity measurement (AIM) with regard to validity as well as temporal and economic efficacy. While the informative value of the three methods correlated significantly, the efficacy of the IgG WB dominated.
RESUMO
BACKGROUND: In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood. METHODS: To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1-/- mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion. RESULTS: We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion. CONCLUSIONS: Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.
Assuntos
Plaquetas/metabolismo , Encéfalo/patologia , Progressão da Doença , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/patologia , Linfócitos/metabolismo , Animais , Encéfalo/metabolismo , Circulação Cerebrovascular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , RatosRESUMO
PURPOSE: Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. METHODS: Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO. RESULTS: After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1ß significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity. CONCLUSIONS: Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.
Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Animais , Infarto da Artéria Cerebral Média , Inflamassomos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
RATIONALE: Ischemic stroke is a leading cause of morbidity and mortality worldwide. Recanalization of the occluded vessel is essential but not sufficient to guarantee brain salvage. Experimental and clinical data suggest that infarcts often develop further due to a thromboinflammatory process critically involving platelets and T cells, but the underlying mechanisms are unknown. OBJECTIVE: We aimed to determine the role of CD (cluster of differentiation)-84 in acute ischemic stroke after recanalization and to dissect the underlying molecular thromboinflammatory mechanisms. METHODS AND RESULTS: Here, we show that mice lacking CD84-a homophilic immunoreceptor of the SLAM (signaling lymphocyte activation molecule) family-on either platelets or T cells displayed reduced cerebral CD4+ T-cell infiltration and thrombotic activity following experimental stroke resulting in reduced neurological damage. In vitro, platelet-derived soluble CD84 enhanced motility of wild-type but not of Cd84-/- CD4+ T cells suggesting homophilic CD84 interactions to drive this process. Clinically, human arterial blood directly sampled from the ischemic cerebral circulation indicated local shedding of platelet CD84. Moreover, high platelet CD84 expression levels were associated with poor outcome in patients with stroke. CONCLUSIONS: These results establish CD84 as a critical pathogenic effector and thus a potential pharmacological target in ischemic stroke.
Assuntos
Plaquetas/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Inflamação/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , AVC Trombótico/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Coagulação Sanguínea , Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/imunologia , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estudos Prospectivos , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , AVC Trombótico/genética , AVC Trombótico/imunologiaRESUMO
Background and Purpose- The selection of appropriate neurological scores and tests is crucial for the evaluation of stroke consequences. The validity and reliability of neurological deficit scores and tests has repeatedly been questioned in ischemic stroke models in the past. Methods- In 198 male mice exposed to transient intraluminal middle cerebral artery occlusion, we examined the validity and reliability of 11 neurological scores (Bederson score 0-3, Bederson score 0-4, Bederson score 0-5, modified neurological severity [0-14], subjective overall impression [0-10], or simple neurological tests: grip test, latency to move body length test, pole test, wire hanging test, negative geotaxis test, and elevated body swing test) in the acute stroke phase, that is, after 24 hours. Combinations of neurological scores or tests for predicting infarct volume were statistically analyzed. Results- Infarct volume was left skewed (median [Q1-Q3], 78.4 [54.8-101.3] mm3). Among all tests, the Bederson (0-5; r=0.63, P<0.001), modified neurological severity (r=0.80, P<0.001), and subjective overall impression (r=-0.63, P<0.001) scores had the highest test validities, using infarct volume as external reference. Subjective overall impression had the best agreement between 5 raters (Kendall W=0.11, P<0.001). The Bederson (0-5) score discriminated infarct volume in mice with small (≤50 mm3; r=0.33, P=0.027) and large (>50 mm3; r=0.48, P<0.001) brain infarcts, all other tests only in mice with large infarcts. Combining subjective overall impression with Bederson (0-5) score explained 47.6% of the variance of infarct volume. Conclusions- Despite their simplicity, the Bederson (0-5) score, modified neurological severity score, and subjective overall impression have reasonable validity and reliability in the acute stroke phase. The Bederson (0-5) score equally distinguishes infarct volume in small and large infarcts. Visual Overview- An online visual overview is available for this article.
Assuntos
Infarto da Artéria Cerebral Média/complicações , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/etiologia , Exame Neurológico/métodos , Animais , Masculino , Camundongos , Reprodutibilidade dos TestesRESUMO
RATIONALE: Giant cell arteritis (GCA) is known to present with typical manifestations like temporal headache and visual abnormalities. However, several cases with atypical manifestations were reported. Stroke occurs in 3% to 7% of patients with GCA. PATIENT CONCERNS: A 67-year-old male patient with known hypertension presented with somnolence, disorientation and mild bilateral limb ataxia. The magnetic resonance imaging showed multiple acute infarctions in the territory of the vertebrobasilar system with occlusion of the left vertebral artery. DIAGNOSIS: Ten months later, during a routine neurovascular follow-up, recanalization of the left vertebral artery was observed and a hypoechoic concentric "halo" sign around both vertebral arteries, mainly on the left side was evident. On further examination of the superficial temporal artery, a hypoechoic concentric "halo" sign was also found, which-along with increased inflammatory markers-raised suspicion about GCA. Classical GCA features like headache, temporal tenderness or amaurosis fugax were not present. Repeated in-depth diagnostic work-up including 48âhours Holter-ECG did not reveal another stroke etiology. INTERVENTIONS: Intravenous Methylprednisolone 250âmg/d was immediately started and after 6 days the dose was tapered to 80âmg/d. The patient was discharged on a tapering scheme with the recommendation to start azathioprine. Additionally, we placed the patient on acetylsalicylic acid 100âmg/d and clopidogrel 75âmg/d. However, the patient was not compliant to treatment; he stopped prednisolone early and did not start azathioprine. OUTCOMES: The inflammatory markers were markedly reduced at the beginning of the treatment. After stopping the immunosuppressive medications, the inflammatory markers were once again increased. Three months later, the patient developed bilateral middle cerebral artery and right occipital lobe infarctions. LESSONS: In patients with cryptogenic vertebrobasilar strokes, GCA may be considered in the differential diagnosis, especially if the inflammatory markers are increased.
Assuntos
Arterite de Células Gigantes/complicações , Acidente Vascular Cerebral/complicações , Artéria Vertebral/patologia , Idoso , Biomarcadores , Arterite de Células Gigantes/tratamento farmacológico , Humanos , Mediadores da Inflamação/metabolismo , Imageamento por Ressonância Magnética , Masculino , Metilprednisolona/uso terapêuticoRESUMO
Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2ß1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2ß1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2ß1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2-/- mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2ß1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2ß1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.
Assuntos
Anticorpos/uso terapêutico , Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Integrina alfa2beta1/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Substâncias Protetoras/uso terapêutico , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Colágeno/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/uso terapêutico , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controleRESUMO
Background and Purpose- Acetylsalicylic acid and clopidogrel are the 2 main antithrombotic drugs for secondary prevention in patients with ischemic stroke (IS) without indication for anticoagulation. Because of their limited efficacy and potential side effects, novel antiplatelet agents are urgently needed. Cilostazol, a specific phosphodiesterase (PDE)-3 inhibitor, protected from IS in clinical studies comprising mainly Asian populations. Nevertheless, the detailed mechanistic role of PDE-3 inhibitors in IS pathophysiology is hardly understood. In this project, we analyzed the efficacy and pathophysiologic mechanisms of a novel and only recently described PDE-3 inhibitor (substance V) in a mouse model of focal cerebral ischemia. Methods- Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in 6- to 8-week-old male C57Bl/6 wild-type mice receiving substance V or vehicle 1 hour after ischemia induction. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, as well as the extent of local inflammatory response and cell death, was determined. Results- Inhibition of PDE-3 by pharmacological blockade with substance V significantly reduced infarct volumes and improved neurological outcome on day 1 and 7 after experimental cerebral ischemia. Reduced blood-brain barrier damage, attenuated brain tissue inflammation, and decreased local cell death could be identified as potential mechanisms. PDE-3 inhibitor treatment did neither increase the number of intracerebral hemorrhages nor affect platelet function. Conclusions- The novel PDE-3 inhibitor substance V protected mice from IS independent from platelet function. Pharmaceutical inactivation of PDE-3 might become a promising therapeutic approach to combat IS via inhibition of thromboinflammatory mechanisms and stabilization of the blood-brain barrier.
Assuntos
Plaquetas/metabolismo , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Cilostazol/farmacologia , Inibidores da Fosfodiesterase 3/farmacologia , Acidente Vascular Cerebral/prevenção & controle , Animais , Plaquetas/patologia , Barreira Hematoencefálica/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
In acute ischemic stroke (AIS), there is an alarming discrepancy between recanalization rates of up to 70% by combined recombinant tissue-type plasminogen activator (rt-PA) therapy and mechanical thrombectomy, and no clinical benefit in at least every second stroke patient. This is partly due to ischemia/reperfusion (I/R) injury. In a translational approach, we used mice lacking dense- (Unc13d-/-) or α-granules (Nbeal2-/-) and mice after blocking of platelet glycoprotein receptor (GP) Ib conferring protection from I/R injury. These mice underwent transient middle cerebral artery occlusion (tMCAO) and, as in the clinic, were treated with rt-PA. Our data show that rt-PA treatment is still safe in conjunction with selected anti-platelet therapies and pave the way for eagerly awaited additive treatment options in acute human stroke.
Assuntos
Fibrinolíticos/uso terapêutico , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Acidente Vascular Cerebral/terapia , Trombectomia/métodos , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Isquemia Encefálica/complicações , Modelos Animais de Doenças , Hemoglobinas/metabolismo , Hemorragias Intracranianas/etiologia , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Estatísticas não Paramétricas , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/etiologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVE: Cardiac diseases are established risk factors for ischemic stroke incidence and severity. Conversely, there is increasing evidence that brain ischemia can cause cardiac dysfunction. The mechanisms underlying this neurogenic heart disease are incompletely understood. Although it is established that ischemic stroke is associated with cardiac arrhythmias, myocardial damage, elevated cardiac enzymes, and plasma catecholamines in the acute phase, nothing is known about the delayed consequences of ischemic stroke on cardiovascular function. METHODS: To determine the long-term cardiac consequences of a focal cerebral ischemia, we subjected young and aged mice to a 30-minute transient middle cerebral artery occlusion and analyzed cardiac function by serial transthoracic echocardiography and hemodynamic measurements up to week 8 after surgery. Finally, animals were treated with metoprolol to evaluate a pharmacologic treatment option to prevent the development of heart failure. RESULTS: Focal cerebral ischemia induced a long-term cardiac dysfunction with a reduction in left ventricular ejection fraction and an increase in left ventricular volumes; this development was associated with higher peripheral sympathetic activity. Metoprolol treatment prevented the development of chronic cardiac dysfunction by decelerating extracellular cardiac remodeling and inhibiting sympathetic signaling relevant to chronic autonomic dysfunction. INTERPRETATION: Focal cerebral ischemia in mice leads to the development of chronic systolic dysfunction driven by increased sympathetic activity. If these results can be confirmed in a clinical setting, treating physicians should be attentive to clinical signs of heart failure in every patient after ischemic stroke. Therapeutically, the successful ß-blockade with metoprolol in mice could also have future clinical implications. Ann Neurol 2017;82:729-743.
Assuntos
Isquemia Encefálica/fisiopatologia , Hemodinâmica/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Animais , Isquemia Encefálica/sangue , Isquemia Encefálica/complicações , Ecocardiografia , Epinefrina/sangue , Insuficiência Cardíaca/prevenção & controle , Hemodinâmica/efeitos dos fármacos , Hidrocortisona/sangue , Infarto da Artéria Cerebral Média , Masculino , Metoprolol/uso terapêutico , Camundongos , Peptídeo Natriurético Encefálico/sangue , Norepinefrina/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
OBJECTIVE: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury. METHODS: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury. RESULTS: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage. INTERPRETATION: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. Ann Neurol 2016;79:970-982.